DE2911033A1 - METHOD AND DEVICE FOR DETERMINING THE MEMBRANE PROPERTIES OF VESICLES - Google Patents

Description

! The invention relates to a method and an apparatus

ι to determine the membrane properties of vesicles, such as

j e.g. the permeability coefficient for a lipophilic

ι ion, the membrane potential, etc. by placing an electrode

; 5, which is selectively responsive to lipophilic ions.

J The vesicles mentioned in the present invention are

¹ structural members comprising a membrane that supports the

j Permeation of a trapped within the membrane

j allow lipophilic ions through the membrane and a

j 1o liquid capable of doing this, lipophilic ions

j and includes living cells such as human j

j lische cells, animal cells, plant cells, bacterial

! cells, etc., organellas such as mitochondria, chloroplast;

I i

ι etc., and man-made vesicles such as liposomes. I.

! ι

j 15 Lately, much research has been very actively conducted [for the purpose of predicting various diseases ; entities, identification of pathogens, education j

the effect of drugs or pharmaceuticals on biomembranes ^! or carry out the screening of new bioactive substances by looking at 'the various functions of Βίοι'

^! membranes, in particular the transport capacity of biomem-I branes made use of.

The methods used to date to determine the transport capacity of biomembranes are as follows: '" *, 25 When a vesicle is placed in a solution of a substance originally present in a living body, such as glucose, the glucose flows into the vesicle and out of the vesicle and at the same time water flows into the vesicle and out of the vesicle, thereby increasing and decreasing the volume of the vesicle. becomes its permeability for the

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• · »a · i

• * »9 * 4 9 I * · · (III

-5 -

Substance (glucose) detected. In the case of erythrocytes, the permeability of the erythrocyte membrane for the Substance measured by measuring the time it takes for the water to enter the erythrocytes, causing them to break and the solution turns red, i.e. the time it takes j to cause hemolysis. Another method; consists in marking the substance with an isotope and adding the isotope that has passed through the cell membrane determine, thereby maintaining the permeability of the cell membrane for the substance.

The radioisotope method is the most reliable method, but it cannot be used for numerous consecutive j Carry out measurements and the radioactive material is dangerous and must be handled very carefully ' will. Therefore this method is not suitable for measuring many j samples. As the method of determining the change in volume of the cell that results from the flowing in and flowing out of the water, a great influence and j Outflow of substance required, which must be sufficient to deform the membrane, agree through this Method, data obtained very often do not match the data on -j measured by the radioisotope method .; The substances which are used for ordinary determinations of the permeability of vesicles for them are almost inveterate those originally contained in, or under the circumstances, the living cells to be determined in which the cells exist. Furthermore, the usual determination of the permeability of vesicular membranes a mere measurement or determination and the measured values can be used for predicting diseases and the like. Not used.

A device for determining the membrane potential of vesicles is described, for example, in "Membrane Potential Measurement Using Lipophilic Ions for Vesicles Directly, Unmeasurable with Small Electrodes "by Makoto Muratsugu et al in

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Digests of Lectures, Second Symposium on Interaction between Biomembranes and Drugs, 1978. This device uses an electrode that is selectively responsive to lipophilic ions, but this device is only suitable for basic research where it is intended to obtain only membrane potentials from vesicles that have large membrane potentials such as mitochondria, bacteria, and the like to determine the Mfcnfoz: .upotentials with this device consists in that a potential difference Δε between the continuous potentials ^; len an electrode in a solution of lipophilic ions before and after the injection of vesicles into the solution and calculates the membrane potential of the vesicles by inserting the value for the potential difference into a complicated equation value of the volume of the vesicles and j the value of the volume of the vesicles _i suspending solution used. With this method and with this apparatus. be delayed door has the definition and measurement ij to the electrode potential after the injection of Ve- j J 2o is Sikel steadily as the constant potential of the electrode \ j after the injection of the vesicles is jj measured in the solution. In general, it takes a while for the | ί electrode potential becomes steady after the vesicles in |

ί I

the solution has been introduced. Human blood cells in particular have both a low membrane potential and a low permeability coefficient. | Particularly at low temperatures between 0 and 25 C, the permeation rate is low, so that it takes so long or more than 30 minutes for the electrode potential to become steady after the blood cells have been introduced into the solution. This common method of determination takes a long time to measure the electrode potential difference Δ E and involves a series of tedious works to calculate the membrane potential of the vesicles by substituting the potential difference Λ E measured for each sample into a complicated equation as described above measure up. Therefore it is impossible to

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tiale of a large number of specimens in clinical Investigation to determine.

The coefficient of permeability of vesicle membranes for Lipophilic ions are closely related to the fluidity of the membranes of vesicles. The membrane potential is the difference between the potentials on both sides of the vesicle membrane and a physical one Quantity that serves as the driving force for ion passage. The membrane potential and fluidity of vesicles both depend only on the properties of the vesicular membrane and are considered to be the basic factors that determine the Define the transport capacity of vesicle membranes. When a valuable piece of information about the transfer of a substance is obtained through a vesicle membrane, for example, it is possible to foresee various diseases, pathogens to identify, to elucidate the effect of drugs and drugs on biomembranes or to elucidate new bioactive substances!

to look through.

I I

The invention relates to a method and a method for. direct and rapid measurement of the basic factors that make the Define the transport capacity of vesicle membranes. It also relates to a method and an apparatus for determination of the membrane properties of vesicles that make the prediction j of different! Diseases, Identification of Pathogenes or enable the screening of new bioactive substances. The invention also relates to a method and an apparatus for the rapid determination of membrane potential, which is a membrane property of vesicles and / or to determine the permeability coefficient for lipophilic ions, which is another membrane property of Vesicles is. In other words, the invention relates to a method and a device that can be used for determination from only the membrane potential of the vesicular membrane alone or only to determine the permeability coefficient the vesicular membrane for lipophilic ions alone or

909840/068

· ■■■■■ · # I ₄ I

■ 'ι < ι ι I · · e »· a, _ - _. Bj

can use for both determinations at the same time, The Method and device make it possible to determine the membrane properties of vesicles at any time during lipophilic Ions flow into and out of the vesicles., To determine.

The invention also makes it possible by the method and the device of the determination of the membrane properties of Vesicles to determine a large number of samples with one device in a short time. After all it is possible to determine the membrane properties of Vesikelix with low membrane potential.

According to the present invention is by one in a Liquid containing lipophilic ions and suspended vesicles j, immersed electrode selective for lipophilic | ! Ions responding to the rate of change in lipo-; • 15 phile ion concentration over time based on the! Presence of the vesicle, as the rate of change of the potential of the electrode measured over time. That - means that vesicles or their suspension in a solution be poured by lipophilic ions or vice versa, in both cases the lipophilic ions flow into the vesicles, thereby causing a change in the concentration of lipophilic ions in the solution. This change is going through measured the electrode that responds selectively to lipophilic ions. The permeability properties of the vesicle membrane for lipophilic ions and / or the membrane potential of the vesicle membrane is obtained by an operation which on the speed of the measured change in potential over time, the volume of the lipophilic ion solution, the Total volume and total surface area of the vesicles suspended in the solution and a temperature Alternatively, the speed of the measured change in potential with time can be determined by the difference in the change in electrode potential, which is peculiar to the vesicle, divided and the value obtained as the index of permeability used by vesicle membranes for lipophilic ions

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I '
J To j 15th be, t • · · t ·· · ···
»» · ■ ·. t
I 1 »·· flt« ι « _t
ι lt · · · a _v Fig. 3 is a schematic diagram showing an example εί S.
£ -9- 2911033: a device according to the invention for the I. "During the measurement of the electrode potential Implementation of the method according to the invention I. preferably a vessel containing the solutions on one shows. Arithmetic unit to the device of FIG. 1, t kept constant temperature. is a schematic diagram following another is coupled. I.
I. I. 2o The drawings are explained below. Example according to the invention shows in which a I ' ⁵ I. Fig. 1 I.
is a flow sheet showing an example of a reference j I. Program of the computing unit (37) shows. I. S. is a graph showing the change j I.
I. changes in the electrode potential, measured with the S. 25th Fig. 2 Method and device of the present invention j dung in connection with erythrocytes from ver i shows different animals. ! is a graph showing the values of the ! Fig. 3 Equation (7) and described below 3o Changes in electrode potential shows when I. Fig. 4 the membrane potential and the permeability I. coefficient for lipophilic ions (tetraphenyl- 1 I.
I. 1
P.
I. Fig. 5 rHig 6 phosphonium) of a human erythrocyte membrane bran according to the method and with the device of the present invention. is a graph showing the result nisse of measurements of the permeability coefficient of liposomes for lipophilic ions (benzyl 909840/0681

■ I II * · at ·· »·

II · r _a

• I t »■ ■ ■» ft

III · · f

Il> · · t ·

-lo-

Fig. 7

phenyldimethylammonium) show by the method and with the device according to the invention have been obtained under different temperature conditions.

is a graph showing the results of the measurement of the permeability coefficient of erythrocytes of rats of different ages for lipophilic ions (tetraphenylphosphonium) shows, according to the method and ^; Apparatus of the present invention and the blood pressures of rats. '

Description of preferred embodiments

Referring to FIG. 1, reference numeral 11 indicates a Vessel containing a solution 12 capable of Dissolve lipophilic ions, with vesicles being suspended in the solution 12. The vessel 11 is preferably treated, e.g. with silicone to keep the vesicles from sticking or sticking to the inner wall of the vessel 11 and preferably the vessel 11 is made of transparent material, to facilitate observation from the outside. A lipophilic ion electrode 13 and a reference electrode 14 are partially inserted into the vessel 11 to be in contact with the vesicle suspension. An injector ; 1 x 5 is provided to transfer vesicles into the vessel 11 in a certain way! Inject volume. The injection device 15 can be a syringe, a pipette or the like. however, it is preferred, for example, to use a microsyringe capable of maintaining a constant volume to inject vesicles.

The solution 12 can be any solvent so long as it is capable of containing a predetermined amount of lipophilic To dissolve ions uniformly. However, since the coefficient of permeability and the membrane potential by the property

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I have a strong influence on the solution, preferably a ί aqueous saline solution is used whose pH value, ionic

The composition and osmotic pressure are properly adjusted to suit the determination. The lipophilic ions to be dissolved in the solvent can be either lipophilic cations or lipophilic anions and the ion concentration is preferably as low as possible: in the range in which the response of the electrode; 13 of the Nernst equation follows. The optimal vesicle concentration for suspending the vesicles can be like; are desired to be selected in accordance with the j membrane potential of the vesicles and the SN ratio of the ver ^; turned measuring device. In general, however, it is desirable that the volume of the vesicles be in the range of 0.015 to 50% of the volume of the suspension. With less

as 0.1%, the change in the electrode potential is small, which leads to reduced measurement accuracy. Also in the case of • more than 50% v / the obtained permeability coefficients are calibrated ^; and the membrane potential from their true values, because! j 2o an increase in the amount of adsorption of the lipophi- i; len ions to the vesicular membrane occurs. Nevertheless, the deviation is smaller than in the case of the known customary measuring methods, so that the volume of the vesicles can in certain cases be chosen outside the above-mentioned range.

The lipophilic ion can be positive or negative. As the lipophilic cation, quaternary cations can be used, which are expressed by the general formula R ₄ X, in which R is a hydrocarbon radical or a halogen

3o ned hydrocarbon radical and X is a nitrogen atom, a phosphorus atom or an arsenic atom. For example R comprises alkyl groups such as methyl, ethyl, propyl, butyl; Aryl groups such as phenyl, tolyl; Aralkyl groups such as benzyl; "Cycloalkyl! Groups such as cyclohexyl groups and their halogen

35 substitution products such as chlorophenyl. As quaternary cations of the above general formula, for example, dibenzyl.-Ιί

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1o

2o

Ί 25

3o

-12-

dimethylammonium, benzylphenyldimethylammonium, tetraphenyl phosphonium, tetraphenylarsonium, etc. can be used.

Organoborate or boron-carbanion cage compounds, which are expressed by the general formula R ' ₄ B ~, in which R' is a hydrocarbon radical or a halogenated hydrocarbon radical, can be used as lipophilic anions. R ¹ includes alkyl groups such as methyl, ethyl, propyl, butyl; Aryl groups such as phenyl, tolyl; Aralkyl groups such as benzyl; Cycloalkyl groups such as cyclohexyl; and their halogen substitution products such as chlorophenyl. As the organoborates represented by the above general formula, borotetraphenyl, borotetratolyl, borotetra- (p-chlorophenyl), etc. can be used. As boron-carbanion cage compounds, dicarbaundecaborane, dicarbadodecaborane and dicarbaheptaborane substituted with a phenyl group such as phenyldicarbaundecaborane and methyldicarbadodecahydroundecaborane can be used.

The lipophilic ion electrode 13 selectively responds to the lipophilic ions present in the suspension 12 and may in some cases be referred to as an organic ion electrode will. If E is the potential difference between the lipophilic ion electrode 13 and the reference electrode 14 and C represent the lipophilic ion concentration of the suspension 12, can be any lipophilic Ion electrode can be used as long as it exhibits electrode characteristics represented by the following equation (the Nernst's equation) in the area of concentration G is given from 5 χ 1o ~ to 1 χ Io M:

E =

ZF

Where <* is an inherent constant for the electrode, T is. _J the absolute temperature of the liquid into which the electric -de is immersed, R is the gas constant, F is the

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«I I 4th

• f <■

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See Faraday's constant and Z is the number of charges of the lipophilic ions. In the lipophilic ion electrode 13 is a polymer film 17, which is sealed therein as a sensitive one Substance containing an ion association of a lipophilic cation and a lipophilic anion at one end of the tube 16 attached, for example made of glass or polymer. The tube 16 contains an aqueous solution in which the desired lipophilic ion in a predetermined concentration is dissolved, i.e. the so-called reference solution 18. In the reference solution 18, one end of the reference electrode 19, e.g. a commercially available silver-silver chloride electrode. The electrode 13 is in the suspension 12 dipped on the polymer film 17 side. The reference electrode 14 may be a silver-silver chloride electrode such as

it is commercially available. |

The device is preferably designed in such a way that the vesicles do not settle in the solution 12 during the measurement. For example, a stirring rotor 21 made of a magnet in the solution 12 in the vessel 11 and an electromagnetic one Stirrer 22, which can rotate at a constant speed, mounted under the vessel 11. Due to the magnetic Attractive force between the electromagnetic stirrer 22 and the rotor 21 becomes the rotor 21 at a constant speed rotates to prevent settling of the vesicles in solution 12. In this case, to the risk of To prevent impairment or destruction of the vesicle by the rotor 21, it is desirable to increase the speed of rotation of the rotor 21 to be kept minimal in the area in which the vesicles do not settle. Since also 'the speed of rotation of the rotor 21 has an effect on the potential of the electrode 13, it is preferred to rotate the rotor 21 at a constant speed.

Since the potential of the electrode varies greatly depending on a change in the measurement temperature, preference is given to ^ place the vessel 11 in a constant temperature bath, for example

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1 «Μ Κ *
• · *

• ■ ·

m λ m

-14-

23 long brought. In Fig. 1, the vessel 11 is in
an opening 3o formed in the center of the constant temperature bath 23. The warm water in bathroom 23 with
constant temperature is circulated by a pump 24,
to keep it at a constant temperature.

In order to achieve high measurement accuracy, it is desirable
that the measurement with temperature changes of + o, - C iurcb-i

~ I

be guided. In the event that one fears that disturbances; by electrostatic induction or by any
1ο other electromagnetic interference with the measuring system
are mixed, the vessel 11 and the electrodes
13 and 14 arranged in a shielded box 25.
Other items that do not have electromagnetic interference

generate can, if necessary, be mounted in the protective box 25 i. The protective box 25 can consist of any i Material be made that electromagnetic waves! does not let through. However, in the case of stirring the! Vesicle suspension 12 with the aid of an electromagnetic stirrer 22, preferably the electromagnetic stirrer 22

to be attached outside the protective box 25 in order to exclude the interference generated by the electromagnetic stirrer 22. In such a case, the part of the | The wall of the protective box 25, which contacts the electromagnetic stirrer 22, is made of a material that allows the magnetic lines of force to pass through, for example aluminum. or stainless steel, so that the rotor 21 in the vessel 11
can be driven. A fastening rod 1o is on
attached to the inside of the upper wall of the protective box 25 and the electrodes 13 and 14 v / ground through the

Rod 1o supported.

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»T

•

To the output potential (output potential) of the lipophilic Ion electrode 13 as an electrical signal, based on the time of the arithmetic unit, is in the Injection device 15 a device 26 for triggering a signal is provided, which is a trigger signal for the Determination of the time of the beginning of the application of the above output potential generated. For example, actuated after completion of the injection of a vesicle-containing sample a projection 28, which on the movable Rod 27 of the injector 15 is attached to a microswitch 26 in the housing of the injector is attached to generate a trigger signal. The generation of the trigger signal can start at the beginning the injection of the vesicles or during the injection. In short, the generation of the trigger serves j signals only to inform the computing unit of the injection of the vesicles into the solution 12.

A voltage signal: indicating the potential difference between of the lipophilic ion electrode 13 and the reference electrode 14 is applied to an impedance converter 31 as required

ι j

! given and the voltage signal between the electrical ' j Figs. 13 and 14 is available is converted to a low impedance output. It is also desirable

't

It is worth providing a voltmeter 2o to read the potential difference between electrodes 13 and 14. It it is advantageous to refer to the potential difference through all of the following operations. From this point of view It is preferred that the gain of the impedance converter 31 is 1. With the present In the invention, it is desirable that the zero point potential has a slight drift (zero point shift) and that the impedance converter 31 has a low noise level (noises) or little interference.

With the device according to the invention, size comes of the signal that one wishes to receive from a

small change in the potential difference between the electrodes 13 and 14, so that the measurement accuracy is enhanced if only the change in the potential difference is enlarged. For this purpose, a suitable DC voltage with the opposite sign to the Output from the impedance converter 31 from a small DC generator 32 in accordance with the detected Potential difference obtained between the pair of electrodes and sent to an adder 33 together with the output signal from the impedance converter 31 given to a certain voltage in the output signal from the impedance converter, 31 repeal. The output signal (output signal) of the adder 33, of which a certain voltage, such as required, has been removed, is transmitted to the recorder 34.

During the measurement or determination, the vesicles are first added to the suspension 12 without lipophilic ions, and then afterwards the potential difference between the electrodes 13j and 14 has reached a constant value, the lipophilic Ions are added to the suspension 12, thereby reducing the concentration of lipophilic ions in the vesicle suspension is changed. Alternatively, after the potential difference between the electrodes 13 and 14, which are in the Suspension 12, which contains both vesicles and lipophilic ions, have been immersed, has become constant, a Solution of lipophilic ions in a concentration different from the suspension mentioned above added to the suspension in order to change the lipophilic ion concentration of the vesicle suspension. The speed the change in the potential difference between the electrodes 13 and 14 with time is measured after the lipophilic ion concentration has been changed abruptly as described above. When the lipophilic ion electrode 13 is immersed in the suspension which does not contain lipophilic ions, there is a possibility that the lipophilic ions entrapped in the polymer film 17

• · i I

come out of it and get into the solution, causing a change in the electrode potential. To this to avoid is preferred the rate of changes in the potential difference between the electrodes 13 and 14 with the time of a change in lipophilic ion concentration of the lipophilic ion solution 12, which by adding vesicles or a vesicle suspension caused, measured.

In the embodiment shown with the apparatus of Fig. 1 measurement, the lipophilic ion solution is stirred at a constant speed to such an extent 12 is preferably \ that the activity of the vesicles will not be affected, while the solution 12 at a suitable desired temperature in the range of O to 100 ° C is kept, which, 15 of the living conditions of the vesicles that are to be measured! is derived, and carry out the following operations after making sure that the potentials' of the electrodes 13 and 14 have become constant. The vesicles or the vesicle suspension are injected into the solution 12 through the injection device 15. The changes in the electrode potentials caused by this injection are recorded. The rate of potential change immediately after the injection of the vesicle suspension is taken as an index for the permeability of the vesicle membrane for the lipophilic ions. Preferably, the ratio of the potential change rate immediately after the injection of the vesicles to the extent of the changes in the potential until the potential becomes constant again after the injection of vesicles is used as the index of the lipophilic ion permeability of vesicle membranes. To calculate this index ¹ , the results recorded by the recorder 34 are read off or the arithmetic unit which is described later is used. The difference between the potential before the suspension of the vesicle and the potential which is at its constant value after the injection of the

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Vesicle is broken is defined as the change in the electrode potential difference that is common to each particular vesicle; kel is inherent. Accordingly, in determining '. for many samples of the same type of vesicle the change in the electrical potential difference, which is measured once, can be used as an index of the permeability for the other measurements.

Fig. 2 shows another example according to the invention, in which the ffombrane properties of vesicles of a Computing unit is calculated from the determined changes in the electrode potential. In Fig. 2 are the Parts which correspond to those in FIG. 1 are denoted by the same reference numerals. The output from the Adder 33 is amplified by a DC amplifier 35 as needed and the amplified output signal is recorded by the recorder 34 and transferred to an A-D converter 36 at the same time. The DC amplifier 35 can be any DC amplifier so long as it has such a gain to give an output suitable for being input to the A-D converter 36 to ground and as long as it has low drift and low noise levels. Recording with the recorder 34 is permitted easy detection of the noise level and drift of the measured potential difference between the electrodes and 14 was reduced or not, i.e., whether the noise level and the drift is within the predetermined limits or not. Εε is also possible as a computing unit 37 to use an analog computer and the determined To feed the electrode potential signal as an analog signal to the arithmetic unit without the specific signal in the To convert digital form. However, when high measurement accuracy is required, a digital arithmetic unit is used

In this case, the analog signal from the DC amplifier 35 is converted into digital form by the A-D converter 36 for input into the arithmetic unit 37 via an input /

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Output (input / output) control interface 38 (control interface) converted. It is desirable that the resolution of the A-D converter 36 is more than 8 bits. The output signal from deitiA-D converter 36 is sent to the computer 37 given at regular time intervals or at programmed intervals such that the converter output signal is delivered first in short time intervals and then gradually in longer time intervals. The output signal from the A-D converter 36 is stored in a memory in the arithmetic unit 37.

If a high measurement accuracy is not required, as mentioned above, the arithmetic unit 37 can be an analog arithmetic unit be. It is also possible to combine an analog computer and a digital computer | use. In the case of the digital computer, an ordinary microcomputer can be used. In the case of measuring the Membrane potential of the vesicles and / or the permeability coefficient of the vesicle! Membrane for the lipophilic ions by the computer 37 / are for the arithmetic operation ι necessary data, that is the data with the exception of the electrical; see signals regarding time and detected ^ Potential outputs from the electrodes, such as the j volume of liquid 12 with those suspended therein Vesicles, the total volume and total surface area of the vesicles suspended in the liquid 12 and / or the temperature of the liquid 12, via the input / output device 39 entered the computer 37.

When a sample that contains vesicles, through the injector 15 is injected into the vessel 11, the electrode potentials found can be in certain Cases change abruptly, which is due to the effect that the lipophilic ion concentration in the solution 12 is diluted by the liquid contained in the sample and by other effects. For example when measuring the membrane properties of erythrocytes

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• ·

ten the electrode potentials when injecting blood into the vessel 11 suddenly drop for the first 1o - 2o seconds and then gradually due to the penetration of the lipophilic ions in the liquid drop into the erythrocytes. Hence the influence of such initial changes of the electrode potentials due to the vesicle injection, it is preferable to use the arithmetic operation then to start after the change in the potential difference between electrodes 13 and 14 only is caused by the permeation of lipophilic ions into the vesicles after the vesicles are in the liquid are suspended, and terminate the arithmetic operation at an appropriate time after making sure that the permeability coefficient and / or the membrane potential converge to certain values. To determine the eye | | bucks the start of the arithmetic operation is the trigger

signal from the trigger signal generator 26 via the input; / Output interface parts 38 to the computer 37 are provided. j

The data output from the data input / output device 39 are the permeability coefficient, the vesicle membrane for the lipophilic ion and / or the membrane potential of the vesicles. However, it is also possible to output part of the data entered into the computer 37 and a parameter which represents the adaptability of the measured values to the theory. The data are output by printing with a printing apparatus or by display output with a CRT display. In addition, the volume of the liquid 12 with the vesicles suspended therein, the total volume and the total surface area of the. vesicles injected into the liquid and / or the temperature of the vessel 11 are entered into the computer 37 by a key input device and stored in it in a memory. It is also possible that different * which store data by so-called Digital distribution (digital switches \ and -. As required - from the computer

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1ο

15th

2ο

- ²¹ -

37 to read off or to save part of the data by keys and the other part by digital distributor.

A description will now be given of an example of an arithmetic operation performed by the calculator 37 in the case of high accuracy measurement. In this example, the permeability coefficient P of the vesicle membrane for the lipophilic ion and the membrane potential ψ of the vesicles are based on the following

^T m

Calculated equations:

ρ -

(1 + ξ ) e ^a + (1 + ζ - V ) b

ζ (b

e ^a ) Λ

( V - 1) ^b - e ζ (b + e ^a )

(D

(2)

where R is a gas constant, F is Faraday's constant, Z is the number of charges of the lipophilic ion, T is the absolute temperature of the liquid 12 in which the vesicles are suspended, e is the base of a natural logarithm, In is an arithmetic variable or a Natural log operand and ξ _{ί $} η and ζ are the quantities defined by the following equations

η = e χ ρ

(3)

7 F *

(4)

(5)

where V is the volume of only the liquid 12, that is the total volume of the vesicles, i.e. the sum of the volumes of the vesicles, V is subtracted from the total volume of the vesicle suspension , A is the total surface area

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area of the vesicles, i.e. the sum of the surface areas of the vesicles, E (O) is the electrode potential that the lipophilic ion concentration in the liquid 12 immediately after the vesicles are suspended therein, but before the lipophilic ions have penetrated the vesicles, ^ and E (t_) is the electrode potential at time t_ of the start of the arithmetic operation, which - as desired - can be determined.

a and b are the most likely values for the coefficients a and b based on the Anne Ims. that y (t) defined by Equation (6) below-SLe is expressed by a straight line represented by Equation (T) below.

y Ct) = I, n {-exp

- ^ 1 (E (I) -Ei

U -

(6)

These εί and t> can be determined, for example, using the least squares method. Since y (t) is calculated from the electrode potential E (t) at an arbitrary moment t according to equation (6), if the values of y (t) at the moments t_, t .., t "..... t _r if the output signal from the AD converter 36 is fed into the computer 37

be represented by y_, y ₁ , y ₂ , y,

"a and Id are calculated according to the following equations:

(«-J - t ₀ - τ) C y. - y)

(8th)

Σ '

- t -Tl ²

Λ

V = y - bT

(9)

90 9840/0681

t.r

* '-ti

• <

wherein

" ¹ D 'ClO)

-__1 "

η + 1 ί-ly j Z ₁₁ \

Next, a description will be given of the process in which equations (1) and (2) are derived. The flow rate 0 (t) of the lipophilic ions flowing through the vesicle menibran is when the lipophilic ion concentration in the vesicles with a potential ψ _η on the inside of the vesicle membrane at a given point in time is t C (t) and the lipophilic ion concentration in of the liquid 12 at a given time tj

ι!

C (t) is obtained by the following equation: j

PZF ^ m C 'Ct) -C ¹¹ Ct) CXp (ZFfci / BT) RT' 1 - exp (ZFiPm / RT)

For the derivation of equation (12), reference is made to the literature _; referenced, e.g., JR Sacks et al in Transport through Red; | Cell Membranes, "The Red Blood Cell" Ed. DM Surgenor, | 2nd Ed., Vol. 2, pp. 613-703, Academic Press, 1975. What is measured by the apparatus of Fig. 2 is the lipophilic ion concentration C (t) in the liquid 12 and the following relationship exists between the . Concentration C (t) and the flow rate 0 (t):

Jj-C ¹ Ct) = - - ^ ^UJ - C ¹³ )

dt V ¹

and the following relationship ^{exists between the concentrations C 1} Ct) and C ¹¹ Ct):

C ¹ O) V ¹ + C ¹¹ O) V = constant = C ^J - (o) V ^J - (lk)

¹ ■ ·

-24-

where C (0) is the lipophilic ion concentration in the liquid 12 prior to the suspension of the vesicles therein. In The following analysis assumes that the moment the vesicles are suspended in the liquid 12, the moment is 0. Taking the flow rate 0 and the concentration C (t) from equations (12), (13) and (14) eliminated, it follows that

dC

{V ^j + V ¹¹ exp (-ZFfra / KT)} c ^! -

PZF ^ m A d t

RTV ¹ V ^U {exp (-ZF '/ m / RT) - 1}

(15)

If one changes the equation (15 J by integrating from the moment t _o to t, the following equation is obtained:

1 + j-exp (-ZFfm / BT)

C ¹ (t) -C ¹ CO)

_V II]

1 + —j-exp (-ZFfm / RT) l · C '(t _Q ) - C' C □)

II

_ ¹¹

r exp ( -ZFWm / RT)

V '

exp C -ZF '^ m / RT) - 1

By introducing the following parameter

exp (-ZF ^ m / RT) ΞΞ X

and substituting equations (3), (5) and (17) into equation (16), the following equation is obtained:

x-i

(18)

»■» ι ι ι

-25-

It has been found through actual measurements that the potential E of the lipophilic ion electrode 13 inserted into the lipophilic solution 12 with concentration C is immersed, is given by the following relationship

RT

(19)

in which the concentration range in which the concentrations C are located between 5 χ 1o M and 1 χ 1o M. a constant inherent in the electrode 13.

If E (O), E (t _n ) and E (t) represent the electrode potentials

I! Which correspond to the lipophilic ion concentrations C (O), C (t) and C (t) in the vesicle suspension 12 in the moments O, t _Q and t, the equation (18)> becomes as follows:

(l + fX) -exp (ZFE (t) / RT) - exp (ZFE (0) / R T ) '(l + £ x) -exp CZ FE (t _o ) / ft T) -exp (ZFE (O 3 / ET)

-) (tt _n )

(20)

If you can find the equation by differentiating its two Pages with respect to time changes and substituting equation (4) into equation (2o) becomes the following equation obtain:

_r ZF dE (t)
■ In C--

<fx

(tt ₀ )

(2X)

The left side of equation (21) agrees with y (t) of equation (6). If you continue to use the appropriate Expressions on the right hand side of equation (21) can be as follows:

I ·

(22)

(23)

For example, equation (21) can be regarded as a linear equation such as equation (7). The left side y (t) of the equation (7) is a quantity obtained from the electrode potential E (t) at a given instant t in accordance is calculated with equation (6) so that when the least squares method is applied to equation (7) is applied, the most likely values ä and ΐ>

to of the coefficients a and b can be obtained with the equations (8) and (9). In addition, if equations (22) and (23) are combined and solved in connection with P and X, and if the relation of equation (17) is used, P and <J / _{m can} be functions of a and b are obtained so that when the most likely values er and b - obtained as I mentioned above - are substituted into them, the permeability coefficient and the membrane potential are obtained as the most likely values as given by equations (1) and j

(2) expressed.

As already defined, E (O) is the electrode potential corresponding to the lipophilic ion concentration in the liquid 12 in the state in which the lipophilic ions have not yet penetrated or incorporated into the vesicles immediately after the vesicles are suspended in the liquid 12 In order to obtain this potential E (O) precisely, the following correction for the electrode potential is made before the vesicles are suspended in the liquid 12. It is the correction for dxt change in the electrode potential caused by the effect of the dilution of the lipophilic ion concentration in the vesicle suspension 12 through the Flüssigke it ver for proper t is the s in the sample t-

~~ 1

«« · Ι · ι ί ■■ · ■ · '

that is injected from the injector 15 and the effect of adsorbing lipophilic ions is maintained the vesicle membranes. To make such a correction the adsorption effect is protected by using a sample identical to the one in the '! injector 15 is contained and in which all vesicles contained therein have burst. The dilution effect will determined by measuring the amount of liquid contained in the sample. But if not so high accuracy is required, the purpose of the measurement may be sufficient! can be achieved even if E (O) is the electrode potential used at the moment when the electrode potential begins to change because of the permeation of the lipophilic

■ change ions in the vesicle.

| 15 The above example of arithmetic operation 'fs j is developed from equation (12). One can also consider in jf j that, as a result of further studies, fj of the transport phenomenon of biomembranes, an equation,; j which represents the flow and further from the equation j! 2o (12) can be derived, recognizing j the structural difference among the vesicles, however; ^; it is unnecessary .to say that can be achieved even in such a case, the purpose% I by appropriate modification arithmetic operation described above dor.

With reference to FIG. 3, a brief description is given of an example of a program for the actual calculation of the permeability coefficient P of the vesicle membranes for the lipophilic ions and / or the membrane potential ψ _{η of} the vesicles. The program is roughly divided into the following 12 parts. In part 1 of IHLg. 3 will be the for di

device 39 read and ξ and C are through the

Arithmetic operation required is calculated from the input / output equations (3) and (4), and the data to be recorded are recorded with the input / output device 39. The data to be entered are the number of loads &

J «X-

-28-

Z of the lipophilic ion, the absolute temperature T of Liquid 12 with the suspended vesicles, the volume

I II

V of the liquid 12 only, the total volume V and the total surface area A of the vesicles in the sample, a size determined by measurement conditions such as a canceled voltage generated by the small voltage generator 32, etc. and the artificial parameters that are necessary for the execution of the program of part 2 and following, such as a time interval Δt. from the injection of the sample to the start of entering the | output by the AD converter 36 into the computer 37, and if subsequent inputs are made at regular time intervals, the time interval ^ t ₂ u; id the time t at the start of the arithmetic operation. However, those of the above data which can be used fixedly, such as the number of charges Z of the lipophilic ion and the artificial parameters, can preferably be seen together with the gas constant R and Faraday's | Constants F are pre-stored in the computer 37. Furthermore, in connection with those data mentioned above, the values of which can easily be obtained from measured values in other ways, such as the total volume V and the total surface area A of the vesicles in the sample to be injected, it is preferred to program the methods of their arithmetic operations and them to be included in Part 1 and the values of the desired data. obtained by entering directly measured values, e.g. the volume of the sample to be injected, the ratio of the volume of the vesicles to that of the sample, and the number of vesicles in the sample. The data to be recorded are the measurement conditions such as the temperature of the liquid to suspend the vesicles therein, etc. and the above-mentioned artificial parameters.

ijA sample is injected into the ! Vessel 11 is injected and at the same time a

■ I till

-29-

Timer started in the computer 37 and at a point in time after a suitable time interval £ t * , which is determined by the input data of part 1, the output from the AD converter 36 is given as VAL (O) in the computer in part 2. O is always an arrangement for storing the values of the various moments in the main data field ARG (O).

After the time interval ^ t ₃ , which is determined by the method entered in Part 1, the output from the AD converter 36 is recorded in the computer 37 and then, after the time interval Δ t ₂ , the output from the AD -Ümsetzer 36 in turn given to the computer 37. These values are successively stored as VAL (D and VAL (2)) in a memory of the computer 37 and at the same time the moments or times of their storage are stored as ARG (I) and ARG (2). After VAL (2 ) the operation goes to part 4, and a differential coefficient is obtained by the forward differential method in accordance with the following equation:

-f 1) - VALf i)

ARG (i

- ARG (i)

in which i = 0, 1, 2, ... The obtained value is stored as DER (i) in a memory. Until the arithmetic operation referred to in part 1 as the starting moment t _Q is reached, it is repeated that the output from the AD converter 36 is stored in order to store its values VAL (i) and the moment ARG (i) in part 3 at the time interval Δ t ₂ and that the differential coefficient in part 4 is obtained.

When the arithmetic operation start moment t _{Q /} determined in part 1 has thus been reached, the output from the AD converter at that time is _{stored as E (t Q} ) in part 5

909840/0681

-3o-

and y \ is calculated according to equation (4). After this
Expiry of the time Δ t ~ , the output of the AD converter 36 is stored in part 3 and the arithmetic operation for the
Differential coefficients is carried out in part 5.
Until a time Kaet (k is an integer which is greater than
the unit is) after the moment t_ has passed,
parts 3 and 4 are repeated and after the expiration
have been time 2k A t ~ be the Differentxalkoeffizienren that ■ / l in time receive 2k ~, Ge in the part 6 'averages. The number of differential coefficients which j are added to calculate the mean value is ι 2k + 1, where k is pre-stored in the computer 37 or entered in ί part 1. At the point in time or moment t + k / it ~ I, the value of the differential coefficient is obtained, which j has been averaged taking into account the values of the previous and ί following differential coefficients,! that is, the shift of the averaged value of the differential '■■ rentialkoeffizienten is set and this value j of the differential coefficient is stored as AVDER (i).
This means that if i _n is taken to be a mini

times an integer that is greater than

- ° -4t

Ό ^t i

the means

value or average value is as follows

i + k

AVDER (i) =

2k + 1 i = i-:

The J)

i = ¹ Q ⁺¹ ' ¹ O ⁺² '

with respect to the fact that the value i is greater than i _Q. In
in this way, the mean value of the Differentialkoeffizien at the time interval Δ t _{o is} obtained.

In part 7, the left hand side y (t) of the linear equation becomes
(7) from the stored VAL (i) and the calculated one

9 0 98 4 0/0681

• is

₍ '' _| ι 'ι I 1 ι ι ι ι ι

-31-

AVDER (i) is calculated according to equation (6) and the result the invoice is saved as Y (I). That means

Y (O = I ^ (VAL CO-VAL (I _n ) ^ nC "U-AVDE R (O]!

λ ι ^u RT ι

in which i = i _Q + 1, i _Q + 2, .... preparations are being made . 5 dripped to the method of least squares in Part 8 \

to apply, i.e. j

, DATAX (x) = ARG (I ₀ + i) - ARG (i _Q + l)

DATAY (x) = y (± ₀ + i)

i -

ι where 1 = 1,2, ...,

! -J ₀ In part ^ 9 the most likely values a (i) j and b (i) until then are obtained by the least squares method j, where the linear equation y (t) = a + b (t - t _Q ) ; is applied to the function y (t). This means that yes (i) and b (i) are obtained according to equations (8) and (9)

ι -g and its program in FORTRAN is as follows: DIMENSION DATAX (300), DATAY (3OO) SUMX = 0
SUMY = 0
N = O

2o SUMXX = 0

SUMXY = 0

The above program is at the beginning of the overall program and the following program is started after the new data have been saved in DATAX (i) and DATAY (i) 25 carried out.

"9WBTOYXrBST

• I Il

• 'I I

• · Il

SUMX = SlMX + DATAX (I)

SUMY ζ = SUMY + DATAY (i)

N = N + 1

AN = N

XM = SUMX / AN

YM = SUMY / AN

SUMX = SUMXX + DATAX (I) ** 2 SUMXY = SUMXY + DATAX (I) * DATAY (I) β = (SUiIXY - SUMX * aUMY / AN) / (SUMXX A = YM - B * XM

- SUMX ** 2 / AN)

Using the above program will be the most likely Values a (i) and la (i) of the coefficient at this moment obtain.

About the adaptability of the measured values to the theory

To check in part 10, the correlation coefficient between the measured value of y (t), ie DATAY (i) and the estimated Werjb, YHAT (i) = £ ₊ £ * DATAx (i) are obtained from y (t). This "FXogram" given in FORTRAN is as follows:

DIMENSION ΥΗΑΤ (3Οθ) = 0

SUMYY = 0
SUMYZ = 0
SUMZZ = 0

The above program is placed at the beginning of the overall program and the following program is after each Storage of the new data in DATAX (i) and DATAY (i) carried out:

909840/0681

-33-

SUMZ = SUMZ + YIlAT (I)

ZM = SUMZ / AN

SUMYY = SUMYY + DATAY (I) ** 2
S ¹ JMXY = SUMYZ + DATAY (l) * yi! AT (l)
SUMZZ = SUMZZ + YHAT (I) ** 2

RYYHAT = (LJUMYZ - SUMY * oUMZ / AN) / ((SUMYY - SUMY ** 2 / AN) * (SUMZZ - SUMZ ** 2 / AN)) ** 0.5

In the above way, the correlation coefficient RYYHAT (i) between the measured value and the estimated value j of y (t) at that moment. |

Part 11 shows the most likely values

P (i) and? Ffii (i) of _the permeability coefficient and the membrane potential at that time are obtained. That is, P (i) and ΨΊη (±) are obtained by adding into equations (1) and (2) the values ζ _u jnd ζ obtained in Part 1, ^ obtained in Part 5

Λ Λ

has been obtained and a (i) and b (i) obtained in Part 9 begins.

In part 12, when the following relationships are obtained among the values of P (i) and ¹ Vm (±) , in part 11, the values of P (i - 1) and ψ _η (± -_ jj are similarly by the immediately above operation in part 11 has been obtained and the predetermined parameters t and c have been set, the obtained RYYHAT (i), P (i) and fm (±) are recorded via the data input / output device 39, and the arithmetic operation is ended.

P (O-

ρΤΠ

iKn (i) - ¹

i - 1)

909840/0691

■ ■ <ι · ι

έ- and t ~ are predetermined parameters that describe the converged states and, it is usually preferred to have values in the range of 0.05 to 0.1. If the required accuracy is 1%, £ ·., And ί ₂ are chosen to be o, o1. If the computed P (i) and Wm (i) have not converged, find that ^! Storage of the output from the AD converter 36 in the time interval & t ₂ , the calculation of the differential coefficient, the averaging of the differential coefficient and the other arithmetic operations for P (D and Wm (i), \ '% as described above take place. |

The program described above is an example of obtaining the permeability coefficient and the membrane potential based on the equations (1) and (2) and is not particularly limited thereto. For example the time interval in which the output of the A-D converter 36 is given to the computer 37 does not always have to be be constant and can be a / suitable orphan programmed time interval. Continue to use the above called the program as E (O) in equation (4) the electrode potential at the moment the electrode! potential begins to change as a result of the permeation of the lipophilic ions into the vesicles. But when

the program is adapted in such a way that E (O) is the potential obtained by correcting | ren for the dilution effect and the adsorption effect in relation to the electrode potential have been made before the suspension of the vesicles, then is it is not necessary to enter the electrode potential into the computer 37 immediately after the injection of the Vesicles to begin as shown in the above program; it is also possible to do the arithmetic operation at any desired time until the electrode

in
potential / has returned to its steady state,

and to achieve the desired goal in the process.

909840/0681

The following describes how the. Method of the invention using the apparatus of the invention, as shown in Fig. 2 is practiced.

First, a power switch (not shown) f »

4 turned on according to the apparatus of Fig. 2, the '

Warm up the apparatus sufficiently. Since the electrode potential in particular is temperature-sensitive, it must be ensured that the temperature of the constant temperature bath 23 is controlled within a permissible temperature range. A certain volume of an aliquot is withdrawn from the liquid which has to meet the necessary conditions to suspend the vesicles and which has the lipophilic ions dissolved therein in a concentration as low as possible in a range in which the response! the electrode on the lipophilic ions of the Nernst's! Equation follows and the aliquot is added to vessel 11. The one that responds selectively to lipophilic ions! f electrode 13 and the reference electrode 14 are immersed in the j 2o liquid 12 and the potential difference between the two electrodes 13 and 14 is determined by the; Read the voltmeter 2o connected to the output side of the impedance converter 31. A voltage which is essentially equal in magnitude to the voltage read 25 and opposite in sign,! is generated by the small DC voltage generator 32 ■ and this voltage is generated by the adder! device 33 is added to the output from the impedance converter 31, ί

to obtain a signal whose voltage is nullified by a certain amount. This signal is generated by the DC amplifier 35 amplified and fed to the recorder 34. It is: desirable, a suitable noise filter or to arrange noise level filters on the output side of the amplifier 35 in order to reduce the disturbances in the output of the DC power amplifier 35 to reduce. After this by recording the recorder 34 it has been ensured that the drift of the output from the amplifier

909840/0681

1o

2o 25th

3o

-36-

45 has been reduced so as to be within a predetermined range, those for the above Data necessary for arithmetic operation, with the exception of the output from the A-D converter 36, via the data input / output device 39 entered the computer 37.

After entering the data is finished, a certain volume of the vesicle is determined for each Purpose is set, injected by means of the injection device 15 into the vessel 11. If a suitable Time period which is pre-stored in the computer 37 has passed after the termination of the injection, is the output from the A-D converter 36 via the input / output control interface 38 given into the computer 37 at suitable time intervals and the arithmetic operation started to measure the membrane potential and the coefficient of permeability of the vesicle for the lipophilic To get ions. After it has been ensured that the values for gene permeability coefficients or the values for the membrane potential converge to certain values, the computer 37 causes the data

input / output device 39 either the membrane potentiometer-j tial and / or the permeability coefficient of the vesicle membrane for the lipophilic ions and then the calculation stops to be ready for the next measurement. The time for a measurement is approximately 10 minutes.

jjTo shorten the time for the measurement, it is preferable to arrange several arrangements of electrodes 13 and 14, -of liquid 12 and of vessels 11, so that the 'Delivery from these electrodes alternately with use a changeover switch can be measured. -That means that while measuring with an electrode \ pair the other electrode pairs are all immersed in the liquid '12 and the subsequent measurement is immediate after the previous measurement with the electrode

909840/0631

1o

15th

2o

25th

3o

• III ι ι

-37-

couple can then be started, the potential of which has become constant, whereby the measurement time is shortened.

As described above, it is possible according to the invention, the basic factors that define the transport capacity of vesicle membranes, directly and quickly measure up.

The invention is further illustrated below with the aid of examples. The examples show the effect of the Invention and that the prediction of pathogens using the method and apparatus of the present Invention obtained membrane properties is possible.

example 1

Benzylphenyldimethylammonium (hereinafter referred to as BPDMA) has been recognized as a lipophilic ion and tetraphenylboron! anion (tetraphenylborate) was chosen as an ion exchanger. Their ion association is sealed in a plasticized vinyl chloride film 0.2 mm thick in order to produce the film 17 which is selectively responsive to BPDMA ⁺ . A measuring system as shown in Fig. 1 was set up; the constant temperature bath 23 was kept at 37 ° C. As a liquid for suspending the vesicles, 2 ml of an aqueous solution of 153 mM NaCl, 17% tris (hydroxymethyl) aminomethane (hereinafter referred to as NaCl-Tris), the pH of which was adjusted to 7.4 with hydrochloric acid, was poured into a silicone treated Glass vessel 11 given. After BPDMA had been dissolved in NaCl-Tris in vessel 11 in such a way that the first-mentioned became 10 molar, the solution was left to stand until the electrode potential became constant. Then, erythrocytes conventionally collected from oxenf humans and rats were each injected into the above solution to give 6% hematocrit (blood centrifuge value). The changes in the obtained electrode potential

909840/0681

are shown in Fig. 4, where the abscissa is time, the ordinate is the electrode potential and the curves 41, 42 and 4 3 represent the measured values for the erythrocytes of oxen and humans and rats, respectively.

The value of the rate of change in electrode potential immediately after injecting the red blood cells is caused by the change in potential until the Zi ^ Ktrcdenpotential again becomes constant, divided and used as an index for the permeability of the red blood cells ^ gjnhr-gj ^ for

"Io BPDMA viewed. The index of ox erythrocytes is the smallest, the index of human erythrocytes is greater than that of ox erythrocytes and the index of rat erythrocytes is the largest. According to the literature (L.L.M. Van Deenen and J. De Gier, Lipids of the Red Cell Membrane, in "The Red Blood Cell", ed.

by D.M. Surgenor, 2nd Auil., Academic Press, pp. 147 bis 211, 1975) the order given above agrees with the order of the increases in the degree of unsaturation of fatty acids in membranes of red blood cells from these living things match what an index for the fluidity of the red blood cell membranes is and also agrees with the order of the increase in Permeability of red blood cell membranes for phosphoric acid or glycerol match, according to another Method was measured. The results shown in Fig. 4 are consistent with the general belief that, as the fluidity of biomembranes increases, their permeability also increases.

Example 2

Tetraphenylphosphonium (hereinafter referred to as TPP) was used as the lipophilic ion and a film which is selectively responsive to TPP ^{+ was} prepared in the manner described in Example 1. The measuring system shown in FIG. 1 was used and the constant temperature bath 23 was kept at 16 ° C. As a liquid for sus-

909840/0681

To suspend the vesicles, 1 ml of an ordinary Ringer's solution was placed in a silicone-treated glass vessel and TPP was dissolved in this solution so that

-5
the former became 10 molar, whereupon the solution was left to stand until the electrode potential became constant. About 3 10 lymphocytes were obtained from 5 ml of fresh human blood, for example by the usual Angio-Conray-Ficoll method, as described in the literature "Tissue typing using a routine onj-step lymphocyte separation technique in Brit. J. Haematol. 18 ( 197o) 229-235 by R. Harris and EO Ukaejifo The lymphocytes thus obtained were each in 1 ml of an RMPI 164o solution containing ο.15 mg of phytohemagglutinin P (hereinafter referred to as PHA-P), which is a type of Lectin and a RPMI 164o solution which did not contain PHA-P, suspended, whereupon the suspensions, after they had been left to stand at 37 ° C. for 5 minutes, were centrifuged at 37 ° C. at 100 g for 10 minutes and the supernatants were removed to obtain the lymphocyte samples (hereinafter referred to as Sample E and Sample F)

the samples E and F each in a glass vessel 11 with con | When the electrode potential was injected at a constant electrode potential, the electrode potential fell instantly due to the dilution effect, but then began to fall gradually, and in a period of 2-5 minutes after the injection, the electrode potential fell at a substantially constant rate. The rate of potential decrease in the case of sample E was about 15% greater than in the case of the sample F. It is believed that this phenomenon is caused by the fact that _r when lymphocytes are stimulated by PHA-P, the cell membranes of a Have fluidity that facilitates the flow of lipophilic ions into the lymphocytes. The phenomenon that lymphocytes, which are normally at rest, are stimulated by an antigen stimulation such as, for example, lectins, to transform themselves into blasts for multiplication and differentiation, is called

909840/0681

-4ο-

Appearance of the formation of lymphoblasts called. By the ratio of the formed blasts to all lymphocytes that are stimulated by antigens, is measured, it is possible to determine the degree of recovery or improvement of immunologically monitored patients to diagnose autoimmune diseases diagnose the cause of allergies through drugs to identify and determine adaptability for organ transplants. Such a measurement the ratio of lymphoblasts converted to blasts to all lymphocytes stimulated by antigens has been playing an important role in clinical testing and it has recently been the possibility of applying this measurement to early diagnosis and detection of relapses Cancer reports. It can therefore be assumed that this determination or measurement will become more and more important.

The method used so far to measure the above-mentioned ratio is to measure the lymphocytes Fix by staining, observing them under a microscope and counting the number of lymphocytes that are in blasts converted to counting or the incorporation of 3H-thyidine into deoxyribonucleic acid (hereinafter referred to as DNA designated). However, these methods require a lot of time to include complicated finding Operations such as sterilizing the culture and are difficult to perform automatically and therefore not suitable for handling large numbers of samples as expected in the future.

On the other hand, the phenomenon described with the present example shows that the method and apparatus according to the invention make it possible that one can see the phenomenon of the conversion of lymphocytes into blasts discovered early and that this method and the device in practice for fast measurements of the

ratio of blasts to lymphocytes generated by antigens stimulated can be used.

Example 3

TPP was taken as the lipophilic ion, the measurement system of Fig. 1 was carried out by the same method as described in Example 2 and the temperature of the constant Temperature bath 23 was kept at 37 ° C. Used as a liquid to suspend the cells 2 ml of NaCl-Tris were placed in the silicone-coated glass vessel 11 and TPP was dissolved in the liquid in such a way that

-5
that it was 10 molar, whereupon the solution was left to stand until the electrode potential became constant.

Smooth muscle cells were obtained by the usual method I. from the cerebrovascular walls of stroke susceptible spontaneous hypertensive rats (hereinafter referred to as SHR-SP), 90 days old and from normal pressure rats (Wistar-Kyoto, hereinafter referred to as WK) of the same age extracted. The extracted smooth muscle cells were each in the glass!

Vessel 11 injected. ^, so that the cells made up 5% by volume. The rate of change in electrode potential immediately after smooth muscle cell injection of SHR-SP was 10% greater than that of WK cells. In the literature, A.W. Jones (1973) Circul Res. 35, 563-572 reports that smooth muscle cells of SHR-SP have a higher ion permeability of the cell membrane than in the case of WK cells. That shows that the permeability of the smooth muscle cell membrane according to that described in the present example Method can be measured.

Example 4

In this example, the device according to FIG. 2 and human red blood cells as vesicles are used. Blood (volume en V _g =

909840/0681

! 79 µl, hematocritwer /; Ht - 38, ο %, red blood cell density

ι / r

Nc = 3.89 χ 1o cells / ul blood), from a peripheral Human vein extracted, used.

• Used as a liquid to suspend the vesicle 5 NaCl-Tris in a volume V, of 1.5 ml used.

TPPs whose concentration in the liquid was 12 1o molai: were used as lipophilic ions.

The j Electrode 13 was made in the following manner: 'An ion association of TPP and tetraphenylboron anion' (hereinafter referred to as TP3 ~) was based on the usual i Way made. In a solution in which polyvinyl chloride as polymer, dioctyl phthalate as plasticizer and ' ELmethylformamide as a solvent in one weight -

'15 ratio of 1: 4: 1o were mixed, was said to be Ion association dissolved to give 0.1 wt .-% and a film of 0.2 mm thickness was by the casting method manufactured. A cut-out portion of the film was attached to the top of the plasti- with tetrahydrofuran.

Fastened vinyl chloride tube 16, which has an inner diameter of 4 mm, an outer diameter of 6 mm and a length of 20 mm. A 10 molar TPP® solution was filled into the tube 16 as comparison solution 18 and the tube at the tip of a silver-silver chloride electrode HS-9o7 (Toa Electronics Ltd.) attached and used as the lipophilic ion electrode 13, which responds selectively to TPP. A HS-907 electrode was used as the reference electrode.

A glass tube treated with silicone was used as the vessel 11 with an inner diameter of 15 ml and a height of 40 mm, which was designed in such a way that it was the ends of the lipophilic Ion electrode 13 and the reference electrode 14 could accommodate.

909840/0681

Il Il ι t I

-43-

The other parts of the apparatus were as follows: An electromagnetic stirrer 22 with controlled speed was manufactured by the applicant itself using a Q-con motor from Japan Servo Co.Ltd. j was used. The rotor 21 is an ordinary rotor j 1o ram in length. The constant temperature bath 23 was made by the applicant himself from acrylic resin and had an opening to receive the vessel 11. The circulation pump 24 for the warm constant temperature water was from Haake, Germany. The injector 15 was a 100 ul microsyringe. The protective box 25

/ I

was made from aluminum by the applicant.! The impedance converter 31 was the TR-8651 model of Takeda Riken Co., Ltd., the small DC voltage generator 32 was a model 31o9 made by Yokogawa Electric Viorks, the recorder 34 and the DC amplifier

35 were from Yokogawa 's Electric Works. The AD translator

36 was an 8-bit resolution converter from Burrbrown Inc., the input / output control interface 38 was from Motorola Semiconductor Products Inc.

The data input / output device 39 was a combination an input device with keys and a thermal printer. The arithmetic unit 37 was one Combination of a memory circuit of 32K-byte capacity and a microprocessor. The program was that described in FIG.

The temperature of the constant temperature bath 23 was set at 37 ° C. Since the potential difference between the lipophilic ion electrode 13 and the reference electrode 14 was -184 mV, the voltage was +184 mV generated by the small DC voltage generator 32.

Indönuit to the output side of the DC amplifier 35 a low pass filter that has a resistance of 1o ΚΛ and a tantalum capacitor of 1o nF

909840/0681

1o

15th

2o

25th

(* ι ι t ι

-44-

231.1033

was connected, the noise or interference could be suppressed to less than 10 uV. About 1 hour after switching on the power for the entire apparatus, the drift in the output from the was DC amplifier 35 about + 0.1 mV / hour, so that data has been entered into the computer 37.

The data thus entered were the volume V, (= 1.5 ml) of the liquid 12 for suspending the vesicles, the Volume V (= 79 µl) of the injected blood, the hematocrit value Ht (= 38, ο%) of the injected blood that

Erythrocyte x eight N (= 3.89 χ 1o ~ ⁶ cells / ul blood),

ο '

the temperature T (= 31o, o K) of the constant temperature bath 23 and the canceled voltage SUBPOT (= -184mV) generated by the small voltage generator 32.

The volume V of only the liquid 12 for suspending the erythrocytes and the total volume V and the total surface area A of the injected erythrocytes, which are required for calculating P and ys _m according to equations (1) and (2), were determined by the calculator 37 calculated according to the following equations:

/ T = v _{b +} v _s (i - ^)

, 11

= V

Ht
s'100

A =

x 10 ~ ⁹ - |

- ·) ^J. (V -N

S C

The equation for calculating A was set up assuming that the volume and surface r: ^ area of a standard human erythrocyte 9o And that the erythrocytes, although of different sizes, are similar in shape.

909840/0681

-45-

The other I parameters required for the calculation were all stored in the computer 37. The values of the main parameters are as follows: The time interval Λ t .. between the injection of the sample and the start of the input of the output from the AD converter 36 into the computer 37 is 15 seconds. The time interval Δ t "for entering the following output from the AD converter 36 into the computer 37 is 6. o seconds. The point of time or the moment t _{Q /} at which the arithmetic operation begins is 51 seconds. The number of moments k shifting before and after the current moment based on time when the mean shift value of the differential coefficient is obtained is 6. The parameter L ₁ necessary for stopping the calculation is

⁼ o, o1.

When human blood in the vessel 11 after completion the input of the above-mentioned data had been injected, the computer 37 began the output from the A-D converter 36 from the specified moment and the computer 37 performed the calculation at each moment and wrote out the calculated results.

Fig. 5 shows curves obtained by plotting the calculation results. The black points are the results obtained by plotting against the moments DATAX (i) of the values DATAY (i) of y (t) of the moment DATAX (i) defined by equation (6) after starting the calculation . The solid line 44 is a straight line y (t) = % + "b (t - t) (at this time a = -4, o2 and Id = -6, 399). The broken line 45 shows the line through the Changes in electrical potential were recorded on recorder 34. The calculation was stopped every 7 minutes after the start of the calculation, and the final calculation in this case resulted in the following:

909840/0681

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2311Q3a

potential

of human erythrocytes is -3o.8 mV>

the permeability coefficient P for human erythrocytes for TPP is 7.19 χ 10 cm / sec. and the correla

tion coefficient b

RYYHAT between y (t) and y (t) = a +

bt is o.993. As shown in Fig. 5 and the value for RYYHAT, the values for y (t) of the equation (6) _f plotted for tt _{o are} almost a barley line, which guarantees the measuring method of the invention. About it hj.na_j. the membrane potential and the permeability coefficient of human erythrocytes compared to TPP are correct

Tritiated TPP.

, measured by the radioisotope method, with with

good with the above values

With the usual Membranmeßmethoden the operation can v / ground j is not performed before the electrode potentials are constant after the blood is injected and this time period may be relatively short, because the overall \ speed of permeation of lipophilic ions through the red cell membranes at a as high as 37 ° C, but more than 15 minutes j are required. In addition, it is necessary to do the tedious; continue to perform the calculation by turning the \ variation of the electrode potential into a complicated! Equation uses and only receives the membrane potential, j On the other hand, the method according to the invention j has the special effect that not only the values of Mem- j

branpotentials, but also the permeability coefficient can be obtained simultaneously in a short time of about 7 minutes. Hence the invention makes a great contribution to medical research and accelerates clinical trials.

Example 5

This example was carried out under the same conditions as Example 4 with the exception that Liposomes as vesicles and BPDMA as lipophilic ions

_ 909840/0681

iii ^ JH1 WtnVWtfWffiffiWff ^ t ^^ ttWBW ^ ffW ^ I ^

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were used. Two types of liposomes were used after made in the usual way. One contained only dimiristoylphophatidylcholine (hereinafter referred to as liposome A) and the other contained dimiristoylphosphatidylcholine and cholesterol (hereinafter referred to as liposome B j). When the liquid in the liposomes was! a 154 mM sodium citrate solution with a reduced to 7.4 r set pH used. The molar ratio of chole- ' sterol in the lipid of liposome B was 37.5%. The permeability coefficient the liposomes A and B for BPDMA were measured under different temperature conditions in the same manner as described in Example 4. The measured results against the temperature change are shown in FIG. Black circles mean the measured values for that Liposome A and white circles denote the measured values for liposome B. FIG. 6 shows that the in this Example liposomes used also have the double effect of cholesterol, i.e. the effect that the Adding cholesterol to the fluidity of a phospholipid; membrane at temperatures above the phase transition point (near 20 ° C in this example) of the Phospholipids are reduced and the fluidity is increased at temperatures below the phase transition point. So allows the use of the device according to the invention to easily determine the temperature dependencies of the permeability coefficient of vesicle membranes for lipophilic ions (corresponding to the fluidity of the vesicle membrane), which makes it possible to easily valuable; Obtain information for general medicine and clinical medicine, such as the phase transition point the vesicle membrane, etc. This is a great contribution to the advancement of medicine.

Example 6

Measurements were carried out in the same manner as described in Example 4 in order to determine the permeability coefficient

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2311QΪ3

th for lipophilic ions (TPP) in erythrocytes, which according to the usual method once from to Stroke-prone spontaneously hypertensive rats that were different days of age (SHR-SP) and normal intense rats (WK) that were also different days old. The measurement results are shown in FIG. 7 along with changes in blood pressure with age. The measurements were made at 17.5 ° C carried out. Black circles show the measured values of the permeability coefficients of the red blood cell membranes of the SHR-SP rats and white circles the values for WK rats, in each case against TPP, plotted versus the age in days of rats. Black triangles show the measured blood pressure values of the SHR-SP rats and white triangles show the measured blood pressure values of the WK rats, in each case plotted against the age of the rats in days. The permeability coefficient of the red blood cell membrane of the SHR-SP rats versus TPP is significantly greater than that of WK rats before blood pressure rises. This indicates that the prediction of genes for the stroke or the sdilagan attack by the method and device according to of the present invention can be taken before blood pressure rises.

9 * 09840/06

Claims (1)

D-5000 COLOGNE 1 March 1-2o, 1979 AvK / IM Method and device for determining the membrane properties of vesicles Claims 1 J Method for measuring the membrane properties of vesicles in which a liquid is lipophilic Ions are dissolved and vesicles are suspended, an electrode that responds selectively to lipophilic ions is immersed is, in order to determine their potential, whereby the membrane properties of the vesicles are measured, characterized in, that the time course of the change in the lipophilic ion concentration of the liquid, based on the presence the vesicle, measured as a change in the electrode potential and that the rate at which it moves changes per time, is obtained as a measurement signal and thus the membrane properties of the vesicles are measured. 2. Method according to "Claim 1, characterized in that the change in the lipophilic ion concentrate over time by adding the vesicles or versicles suspended in a liquid to the solution in which the lipophilic Ions are dissolved, is caused. 909840 / 0S81 2311033 .3. Method according to claim 1, characterized in that the change in the lipophilic ion concentration over time caused only by the permeation of the lipophilic ions into the vesicles. 4. The method according to claim 1, characterized in that the changing speed of the determined over time Measurement signal is divided by a difference in electrode potential that is inherent in the vesicles. 5. The method according to claim 1, characterized in that the permeability coefficient of the vesicle! Membrane for the Ii- 'phile ions and / or the membrane potential of the vesicle membrane is calculated from the time change in the speed of the test i provided measurement signal. ; 6. The method according to claim 1, characterized in that the measurement is carried out while the liquid is at is kept at a constant temperature. 7. Device for determining the membrane properties of vesicles, comprising a vessel for the lipophilic ions and the vesicles and an electrode which is selective for lipophilic ions responds to phile ions, characterized in that they are a / Computing unit (37) which is electrically connected to the electrode (13) is connected to the change in speed over time to calculate the electrode output power. 8. The device according to claim 7, characterized in that it has an injection device (15) for injecting a constant volume of the vesicle or the lipophilic ion solution in the vessel (11). 9. Apparatus according to claim 8, characterized in that that a device for triggering a signal (26) is provided, which the arithmetic unit (37) an electrical signal transmitted that with the injection of the injection 909840/0681 ~ ³ ~ 2811033 (15) is synchronized. To. Device according to claim 7, characterized in that!
that they have a constant temperature bath (23) for the vessel
(11). 11. The device according to claim 7, characterized in that
that the vessel (11) and the electrodes (13, 14) in one
shielded box (25), the electromagnetic interference
prevents, are arranged. 12. The device according to claim 7, characterized in that
that the arithmetic unit (37) calculates the volume of the solution, the total volume and the total surface area of the vesicles j I suspended in the solution, and the temperature of the j | Solution contains stored, and the permeability coefficient- | ten of the vesicle membrane for the lipophilic ions and / or the membrane potential of the vesicle membrane is calculated. | 13. The device according to claim 7, characterized in that! | that it has a recorder to which the ι | detected measurement signal is transmitted for recording. I f 909840Λ0681